Distance and density dependence in two native Bornean dipterocarp species

Abstract The Janzen–Connell hypothesis proposes that density and distance‐dependent mortality generated by specialist natural enemies prevent competitive dominance. Much literature on Janzen–Connell mechanisms comes from the neotropics, and evidence of the role of distance and density‐dependence is still relatively sparse. We tested the predictions of the Janzen–Connell hypothesis in a South‐East Asian system dominated by mast fruiting species. We hypothesized that seedling survival would decrease with distance and density, seedling growth would increase, and herbivory would decrease, according to the predictions of the Janzen–Connell hypothesis. Experiments were conducted to determine the strength of the Janzen–Connell mechanism by manipulating the density and identity of tree species as a function of the distance from parent trees. Survival of conspecific seedlings was reduced near adult trees of one species, but not another. High densities of seedlings decreased the growth of conspecific seedlings of both species. In both species, herbivory rates decreased with distance in low‐density areas. This study indicates that dipterocarp species experienced weak Janzen–Connell effects of distance and density dependence at the growth stage studied. Future studies in this system might focus on earlier life‐history stages such as seeds and small seedlings, as well as studying mortality during mast‐seeding events.


| INTRODUC TI ON
Tropical rainforests are the most diverse ecosystems in terms of community structure and species diversity (Chazdon, 2003;Edwards et al., 2014;Gardner et al., 2009;Steege et al., 2015). It has been a challenge for ecologists to understand the process that maintains diversity in plant communities, and this is especially true in hyperdiverse tropical forests (Bagchi et al., 2014;Chesson, 2000;Dalling et al., 1998;Steege et al., 2015;Terborgh, 2012). In tropical forests the number of species appears to greatly exceed the number of limiting resources (Hutchinson, 1961). Under such circumstances the competitive exclusion principle predicts that the superior species will drive other species to extinction (Hardin, 1960;Levin, 1970).
Various hypotheses have been proposed to explain the diversity of tropical forests (Connell, 1971;Connell, 1978;Hubbell, 2001;Janzen, 1970;Schoener, 1974). Niche partitioning is a mechanism that explains high diversity through minimizing competition between species (Schoener, 1974). Kraft et al. (2008) showed that niche partitioning can contribute to the maintenance of forest diversity, and other studies have indicated that there is niche partitioning in dipterocarp forests (Gunatilleke et al., 2006;Potts et al., 2002).
One of the leading theories for explaining tropical forest diversity is the Janzen-Connell hypothesis (Connell, 1971;Janzen, 1970).
The Janzen-Connell hypothesis suggests that specialized natural enemies (pathogens, seed predators, and herbivores) play a vital role in maintaining the diversity of tropical plant species in a densitydependent manner. This works through reducing the survival of seeds and seedlings near conspecific adults where seed density is the highest. According to this hypothesis, if natural enemies are sufficiently specialized, they aggregate on high densities of seeds or seedlings of their hosts close to adult trees (Dalling et al., 1998;Freckleton & Lewis, 2006;Fukue et al., 2007;Hülsmann et al., 2021;Swamy & Terborgh, 2010;Traveset, 1990). The density-dependent nature of the resultant mortality prevents competitive exclusion (Comita & Stump, 2020). This is because locally abundant species will experience higher mortality than rare ones, thus allowing the rarer species to survive and coexist. This density-dependence acts as a stabilizing mechanism that can promote the maintenance of diversity (Chesson, 2000).
This geographic gap is important to address because, globally, forests differ from each other in important ways. For example, Asian dipterocarps are unique for their mast reproduction and fruiting. In Southeast Asian forests, the dominant Dipterocarp species are usually involved in community wide mast fruiting events (Appanah, 1993;Ashton, 1988). It has been hypothesized that systems undergoing mast-fruiting may not experience strong density and distance-dependent predation because of predator satiation (Cannon et al., 2021;Curran & Webb, 2000;Webb & Peart, 1999). This is because all species produce large numbers of seeds simultaneously, and there will be insufficient predators to generate significant mortality.
Several studies have found that predator satiation, especially in Dipterocarps, weakens the Janzen-Connell mechanism (Ashton, 1988;Curran & Webb, 2000;Paoli et al., 2006). Several characteristics of Dipterocarp seeds and seedlings such as large size, poor chemical defense, and being energy rich make them attractive food for wild pigs, Sus barbatus (Ashton, 1988;Curran & Webb, 2000), and weevil beetles, family: Curculionidae (Bagchi et al., 2011;Lyal & Curran, 2000). From the perspective of maintaining diversity, generalist natural enemies are expected to have a low diversity-enhancing effect compared with specialists (Curran & Leighton, 2000;Freckleton & Lewis, 2006;Gilbert, 2005). Theory suggests that generalist natural enemies should not generate Janzen-Connell mechanisms (Freckleton & Lewis, 2006). More recent work has shown that limited amounts of generalism can nevertheless still yield diversity enhancement (Sedio & Ostling, 2013). Bagchi, Swinfield, et al. (2010); Bagchi, Press, and Scholes (2010) have shown evidence for distance-dependence in dipterocarps, however overall, there is little understanding of the role of Janzen-Connell mechanisms in hyperdiverse forests with mast-seeding.
Here we examine the effect of distance and density on two Bornean dipterocarp species, Parashorea malaanonan and Shorea johorensis. We manipulated the density and type of tree species (Parashorea malaanonan and Shorea johorensis) as a function of the distance from conspecific adult trees. We experimentally tested the strength of Janzen-Connell hypothesis in these two native dipterocarp species, specifically addressing the following hypotheses: (1) the survival of conspecific seedlings will decrease with proximity to conspecific adult trees (distance-dependence) and within high density of conspecific seedlings (density-dependence) compared to heterospecific seedlings; (2) high density of conspecific seedlings will decrease the growth of conspecific seedlings; (3) Herbivory rates in conspecific seedling will decrease with increasing distance from conspecific adult trees; and (4) Leaf herbivory in new leaves decrease with increasing distance from conspecific adult trees.  (Marsh & Greer, 1992). The soils in DVCA are orthic acrisols, developed on sandstone and mudstone. Clay percentage in these soils ranging from 30% to 60% with acidity ranges from 5.3 to 4.0. The mean minimum and maximum temperature at the field center is 22.6 and 31.2°C respectively, while mean annual rainfall is 2881 mm (Walsh et al., 2011).
Our study focuses on two dominant dipterocarps in the region, Parashorea malaanonan and Shorea johorensis. Parashorea malaanonan is one of the native dipterocarp species in this region (18.6 stems/ha) (Stoll & Newbery, 2005). Parashorea malaanonan is classified as White Seraya Light Hardwood and known as a fast-growing dipterocarp species in Borneo (Bagchi, Press, & Scholes, 2010;Bagchi, Swinfield, et al., 2010). Shorea johorensis is native dipterocarp species, fast-growing and big emergent trees that can usually be found in Danum Valley Conservation Area, with 24.6 stems/ha (Brown & Whitmore, 1992;Stoll & Newbery, 2005). It belongs to Light Red Meranti group, and frequently used in plywood and veneer. Following recent community-wide mast fruiting events, seedlings of these two dipterocarp species were easy to locate and are often intermingled.

| Field experiment
Conspecific adult trees of P. malaanonan and S. johorensis were located by searching along a 2 km network of trails adjacent to the field center. These two species were distinguished in the field based on their key characteristics (Soepadmo et al., 2004).
At each conspecific adult tree (diameter at breast height > 30 cm), one transect was set up from 2 m to 30 m away from conspecific adult tree. We checked that there were no adult trees within a distance of 30 m of each parent. Twelve 1 m × 1 m plots (1 m 2 ) were established along each transect, consisting of four experimental plots each at distances 2 m, 15 m, and 30 m from the conspecific adult tree, respectively (following Bagchi, Swinfield, et al., 2010;Bagchi, Press, & Scholes, 2010). Each plot was randomly assigned to one of four treatments: (1) low density of seedlings (4 seedlings m −2 ), (2) high density of seedlings (12 seedlings m −2 ), (3) mixed species with low density of seedlings, and (4) mixed species with high density of seedlings. The above setup was replicated for 10 trees of each species (i.e., 240 quadrats were established in total for both species).
Seedlings of the two species were obtained from the Innoprise-

FACE Foundation Rainforest Rehabilitation Project (INFAPRO) nurs-
ery, near Danum Valley Field Centre. Currently, this nursery has stocks of 28 native dipterocarps species and six other indigenous species. All the dipterocarp seedlings in this nursery are collected from recent mast fruiting events. Germinated seeds of the twostudy species were planted in polybags in July 2014 and kept in the nursery: thus, the seedlings used in this study were 2 years old.
In all experimental plots, existing plants were removed but leaf litter on the ground was left. Seedlings were planted using a planting bar. This is used to prepare holes for seedlings planting. Planting bars provide suitable holes for small seedlings particularly in small plots and prevent excessive disturbance to the forest soil. In total across all treatments 96 seedlings were planted in 12 plots (four plots for each distance) adjacent to each conspecific adult tree.

| Measurements
All seedlings were tagged with numbered aluminum labels and identified to species (or to the lowest taxonomic level possible) with the help of a botanist. The heights of all seedlings were measured by using a 1 m rule. Seedlings height were measured at the beginning of the experiment and at the end of the experiment. Stem diameters were measured just below the cotyledon scar using a digital vernier caliper (Haase, 2008). All leaves surviving from the first census and new leaves produced during the interval were recorded for each seedling.
In order to estimate the rate of herbivory, five leaves were selected from each seedling and labeled with a unique number written in permanent ink on the underside of leaves during the first census (July 2016). Visual estimates were employed in this study where herbivory damage is estimated as the percentage of leaf surface area removed (Stotz et al., 2000). All seedlings were re-measured in June 2017. The number of marked leaves missing, and herbivory of new leaves also were recorded. In each plot, a spherical densiometer was used to determine canopy openness and light availability to seedlings (Lemmon, 1956).

| Statistical analyses
The survival and growth data were analyzed separately for each of the focal species. To test for effects of distance and density treatment on survival of conspecific seedling, seedling data were analyzed using generalized linear models (GLMs) with a quasi-binomial distribution and logit link function (Survival ~ as.factor (Tree) + Distance * Species Identity * Density). The quasi-binomial distribution was used to account for overdispersion. To analyze the effects of distance and density on growth and herbivory of planted seedlings, a linear model was used (Height/Diameter/Herbivory ~ as.factor (Tree) + Distance * Density * Mixture. monoculture). All statistical analyses were conducted in the statistical software environment R version 4.2.0 (R Core Team, 2022).

| Effects of distance and density on survival of seedlings
There was a significant effect of distance from P. malaanonan adult trees on the survival of seedlings (Table 1; F 1, 169 = 9.544, p = .002).
Survival of conspecific and heterospecific seedlings was highest at the furthest distance (30 m) while lowest at the nearest distance (2 m) ( Figure 1a). Closest to the adult (2 m), conspecific seedlings suffer higher mortality compared to heterospecific seedlings in both high-and low-density treatment. However, there was no marginal effect of density on survival when the distance to adults was statistically controlled (Table 1; F 1, 167 = 2.279, p = .133).

| Effects of distance and density on growth of seedlings
Overall, there was evidence that density affected the growth of seedlings, however little evidence of an effect of distance. There was a significant effect of density on height increments of both conspecific seedlings of P. malaanonan (Figure 2a,b, Table 2; F 1, 96 = 4.679, p = .033) and S. johorensis (Figure 2c,d, There was a significant effect of mixture and monoculture planting treatment on diameter increment of conspecific seedlings around trees of P. malaanonan (Figure 3a,b, Table 2; F 1, 103 = 5.438, p = .022). Diameter increment of conspecific seedlings around trees of P. malaanonan increased by 0.6 cm in monoculture plot compared to mixed species planting treatment plot. A weakly significant interaction was observed between the density treatment and the mixed and monoculture planting treatments (Table 2;  increased by 0.51 cm in low-density plot compared to high-density plot. There was a significant interaction between distance and density (F 1, 102 = 4.304, p = .041). Thus, diameter increment increased with distance from adult trees at low, but not high densities (see Table 2).
We found no significant effect of distance and density treatment on number of leaves of conspecific seedlings around both P. malaanonan and S. johorensis adult trees ( F I G U R E 1 Seedling survival at conspecific and heterospecific seedlings as a function of distance from P. malaanonan adult trees at high (a) and low (b) and distance from S. johorensis adult trees at high (c) and low (d) in distancedensity experiment. Error bars represent standard error of the mean after transforming to the proportion scale.

F I G U R E 2
Effects of distance and density on height increment of conspecific seedlings from adult P. malaanonan (a, b) and S. johorensis (c, d) trees with monoculture (a, c) or mixture (b, d) planting treatment. Error bars represent standard error of the mean.

| Effects of distance and density on herbivory
There was a consistent decline in herbivory with distance from parents of both species, as well as evidence for impacts of density as well (Table 3). In the low-density treatment, herbivory rates of P. malaanonan seedlings and S. johorensis decreased with distance from adult P. malaanonan trees (Figure 4) (F 1, 103 = 5.675, p = .019). A significant interaction was observed between distance and density variables (F 1, 103 = 9.165, p = .003), with a negative effect of distance in the low density, but not the high-density treatment (Figure 4a,b).
Around S. johorensis adult trees, there was a significant effect of distance on herbivory rate on S. johorensis seedlings indicating that herbivory rates decreased with distance from adult trees (F 1, 102 = 6.363, p = .013). The herbivory rate of S. johorensis seedlings was negatively affected by seedling density as low density exhibited high herbivory rates compared to high-density plots (F 1, 102 = 7.969, p = .006). Furthermore, mixture and monoculture planting treatment also had a highly significant effect on herbivory rates in S. johorensis seedlings (Figure 4c,d, F 1, 102 = 9.038, p = .003) (see Table 3).

| Effects of distance from parents and density on herbivory of new leaves
There was no significant effect of distance and density on production of new leaves in seedlings of either. We also found no significant effects of distance and density on leaf herbivory in P.
malaanonan seedlings. However, there was a significant positive effect of distance on herbivory of new leaves in S. johorensis seedlings (F 1, 108 = 5.990, p = .016). Effects of distance on herbivory varies significantly between density treatment. Thus, there was a significant interaction between distance and density (F 1, 108 = 4.547, p = .035) (see Table 4).

| DISCUSS ION
Understanding distance and density-dependence in plant communities is essential for understanding species diversity in tropical forests (Liu et al., 2012;Schupp & Jordano, 2011). Our study revealed that the two species exhibited contrasting effects of distance and density-dependence, compared with the predictions of the Janzen-Connell hypothesis. We showed that survival of seedlings located near the adult trees was reduced for P. malaanonan indicating distance dependence occurs, but density dependence does not.
Distance and density dependence were not detected in S. johorensis seedlings. However, we found that in both species, high densities of conspecific seedlings decreased the growth of seedlings.
Moreover, herbivory rates on conspecific seedlings of both species decreased with distance. In addition, our study demonstrated that leaf herbivory for new leaves varies with the distance from the focal adult tree.

| Effect of distance and density on survival
The effect of distance on survival was stronger for conspecific seedlings than heterospecific seedlings around P. malaanonan adult trees, while there was no effect of density on conspecific seedlings. For density-dependence to promote species coexistence, conspecific seedling must be affected more than heterospecif-  Bagchi, Swinfield, et al. (2010); Bagchi, Press, and Scholes (2010) who found that survival of naturally occurring P. malaanonan seedlings suffered greater reductions near conspecific adult trees than heterospecifics.
However, we found contrasting results for conspecific seedlings around S. johorensis conspecific adult trees. In this case the survival of conspecific seedlings is unaffected by either distance or density. Connell (1971) and Janzen (1970) emphasized that natural enemies F I G U R E 3 Effects of distance and density on diameter increment of conspecific seedlings from adult P. malaanonan (a, b) and S. johorensis (c, d) trees with monoculture (a, c) or mixture (b, d) planting treatment. Error bars represent standard error of the mean.

F I G U R E 4
Effects of distance and density on herbivory rates of conspecific seedlings from adult P. malaanonan

| Effect of distance and density on growth
We observed that the negative effect of density on height increment in both P. malaanonan seedlings and S. johorensis seedlings was stronger in high-density treatments compared to low densities. A study by Linkevičius et al. (2014) demonstrated that intense competition can lead to negative effects on height increment in highdensity treatment plots. Such results suggest that when seedlings occur at high densities, intraspecific resource competition and microbe-mediated processes in soils can affect the growth of the seedling, resulting in negative density-dependent processes.
We found that diameter increments of S. johorensis seedlings are highly affected by distance and density. Stoll and Newbery (2005) found that conspecifics seedlings and small trees show decreased growth close to adult conspecific trees in dipterocarps. It is possible that adult trees may take phosphorus from conspecific seedlings that occur near to them via the root system. Several studies have suggested that ectomycorrhizal fungi found in the root system would increase phosphorus uptake from nearby nutrient sources and transfer to their host plants (Brearley, 2012;Perez-Moreno & Read, 2000;Tibbett & Sanders, 2002).

| Effect of distance and density on herbivory
Results from our experiment revealed that there is effect of distance and density on herbivory rate in conspecific seedlings. Distancedependent herbivory rates on conspecific seedlings were observed.
However, no evidence of density dependence was found as herbivory rates in low-density plots are higher than high-density plots.
It is possible because leaf herbivores are satiated with high densities of seedlings (Aide, 1992; Crawley & Long, 1995).

| Further directions
The Janzen-Connell hypothesis suggests that natural enemies such as insect herbivores and pathogens must be host specific.
Host specificity is required to drive Janzen-Connell mechanism in plant communities (Ali & Agrawal, 2012;Clark & Clark, 1984). Dyer et al. (2007) demonstrated that insect herbivores are more specialized in the tropics. However, recent studies found that tropical insect herbivores are more general in their host preferences (Gilbert & Webb, 2007;Novotny & Basset, 2005;Weiblen et al., 2006). In our study, we did not test for the host specificity of natural enemies which is an important element of the Janzen-Connell mechanism.
This could highlight the role of host specificity in maintaining forest diversity. Ghazoul (2016) highlighted that how pathogens may be more critical than insects to maintain distance and density dependence in dipterocarp forests. A recent study by Spear and Broders (2021) showed that generalist pathogens contribute to maintenance of forest diversity in tropical area. Thus, experimental studies on density and distance dependence involving natural enemies host specificity on community level could highlight to what extent that natural enemies can maintain forest diversity.

| CON CLUS ION
Overall, distance and density-dependent effects vary for both species tested in this study. Future studies could consider early life history stages (i.e., seed stage, seed-seedling transition, and young seedlings) and whole-life cycle studies to detect distance and density dependence and their role in maintaining tropical forest diversity. With greater exposure to natural enemies, impacts of distance and density-dependent effects are more likely to increase.

FU N D I N G I N FO R M ATI O N
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data that support the findings of this study such as experimen-